In order to elucidate life phenomena, it is very important to analyze mRNA molecules of diversified genes. By the finding of an RNA-dependent DNA polymerase, in other words a reverse transcriptase, it is made possible to carry out a reverse transcription reaction for synthesizing cDNA with RNA as a template, thereby making remarkable advancements in the method of analyzing mRNA molecules. The method of analyzing mRNA molecules using a reverse transcriptase now has become an essential experimental method in studies concerning genes. PCR method for amplifying DNA fragments with cDNA synthesized by a reverse transcription reaction as a template is referred to as RT-PCR method. The RT-PCR method is utilized in cloning of cDNA derived from mRNA or in the production of cDNA libraries, and also is useful as a method for examining an expression state of a particular RNA.
However, in a case where DNA is contaminated in a sample used in cDNA synthesis, a region on DNA encoding RNA or a pseudogene may be amplified, thereby making it difficult to selectively obtain only the DNA synthesized with RNA as a template. In order to inhibit the production of an amplified product derived from DNA contaminated in the sample, a method in which a nucleotide analog and a compound which lowers a Tm value are used during a reverse transcription reaction (Patent Publication 1), a method including degrading DNA in the sample with an endodeoxyribonuclease (hereinafter, may be referred to as endoDNase) such as deoxyribonuclease I (hereinafter, may be referred to as DNaseI), and thereafter carrying out a reverse transcription reaction, and the like are employed.
In a case where a reverse transcription reaction is carried out after degrading DNA in the sample with DNaseI, in order to inhibit the degradation of cDNA, the DNaseI is generally inactivated or removed prior to the reverse transcription reaction, by a heat treatment, a phenol/chloroform extraction or the like. However, it has been known that the phenol/chloroform extraction is complicated, and that a heat treatment in the presence of divalent metal ions which are essential for the reaction of DNaseI leads to hydrolysis of RNA. Although a method of actions with DNaseI and a reverse transcriptase at the same time is also proposed (Patent Publications 2 and 3), DNaseI would also degrade DNA in a DNA/RNA hybrid, so that the cDNA synthesized by a reverse transcription reaction in this manner is subjected to degradation with DNaseI.